Standard operating procedures

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STANDARDOPERATING PROCEDURES
At the Wellcome Trust Millennium Building

Applied Molecular Genetics Laboratory

Applied Molecular Genetics Laboratory

STANDARDOPERATING PROCEDURES
At the Wellcome Trust Millennium Building

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Wakehurst Place - Millenium Seed Bank Partnership - Applied Molecular Genetics Laboratory

2

Contents

I II III IV V VI VII VIII IX X XI XII

Mini-Extraction Spectrophotometer PCR Electrophoresis Cloning Digest Micro organisms Micro-Centriguges Freeze dryer X-Ray Autoclave Calculations

Wakehurst Place - Millenium Seed Bank Partnership - Applied MolecularGenetics Laboratory

3

STANDARDOPERATING PROCEDURES
Method Code: 01 Subject: Plant genomic DNA mini extractión Section: Research Diagnostics

Toni Devís López - (U.P.V.) Peter E. Toorop - (M.S.B.)

Plant genomic DNA mini-extraction

1. Introduction & Principle
The extraction and isolation procedure we devised consist of the following steps: Disruption of the cellular structure to create alysate. Using the water bath to remove the polysaccharides with the extraction buffer; Separation of the soluble DNA from cell debris and other insoluble material. Purification of the DNA of interest from soluble proteins and other nucleic acids [Organic extraction (with chloroform). Extraction with high-salt (NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remainingpolysaccharides. Removal of RNA by RNase. Precipitation of the DNA in a CTAB-nucleic acid when the salt concentration is reduced by the addition of one volume of 1%CTAB, after 15 min at room temperature. Followed by ethanol precipitation. Rinse the DNA pellet with 70% ethanol].

2. Equipment & Materials
1 M Tris-HCL (pH8) NaCL EDTA CTAB (W/V) DTT Water μL sample 500 0,1 1,4 0,02 2% 0,02 1
STOCK M

23

4

5

6

7

8

9

10

V 0,1 0,467 0,04 P. gr 0,02 μL 50 234 20 0,01 10 186
500

EXTRACTIÓN BUFFER (EB) 100 467 40 0,02 20 373 150 701 60 0,03 30 559 200 934 80 0,04 40 746 250 300 350 400 450 500

1 3 0,5

1168 1401 1635 1868 2102 2335 100 0,05 50 932 120 0,06 60 140 0,07 70 160 0,08 80 180 0,09 90 200 0,1 100

1119 1305 1492 1678 1865

1.000 1.500 2.000 2.500 3.0003.500 4.000 4.500 5.000

PRECIPITATION BUFFER (PB) Tris-HCL (pH8) EDTA CTAB (W/V) Water μL sample 1000 0,05 0,01 1% 1 0,5 0,05 0,02 P. gr μL 50 20 0,01 930
1.000

100 40 0,02

150 60 0,03

200 80 0,04

250 100 0,05

300 120 0,06

350 140 0,07

400 160 0,08

450 180 0,09

500 200 0,1

1860 2790 3720 4650 5580 6510 7440 8370 9300
2.000 3.000 4.000 5.000 6.000 7.000 8.000 9.00010.000

RESUSPENSIÓN BUFFER (RB) NaCL RNAse A
(10μg/ml)

1,5 5

3 μL

0,5

150 5 μL 145
300

300 10 290
600

450 15 435
900

600 20 580
1200

750 25 725
1500

900 30 870
1800

1050 1200 1350 1500 35 40 45 50

Water μL sample 300

1015 1160 1305 1450
2100 2400 2700 3000

TRIS-EDTA BUFFER (TE) Tris-HCL (pH8) EDTA Water μL sample 100 0,01 0,001 1 0,5 0,01 0,002...
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